Working Together to Eradicate TB
4th SA TB Conference 10-13 June 2014 at the ICC Durban.
Selected Abstracts submitted by the CBTBR WITS Node.
Detection of viable but non-culturable bacteria in tuberculous sputum
Melissa Chengalroyen1, Bhavna Gordhan1, Germar Beukes1, Neil Martinson1,2 and Bavesh Kana1
1DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg, South Africa 2Perinatal HIV Research Unit, University of Witwatersrand, Witwatersrand, South Africa
Background: M. tuberculosis, the causative agent of tuberculosis (TB) enters a dormant state allowing it to evade the host’s immune response and reactivate years after primary infection. Resuscitation promoting factors (Rpfs) have been implicated in the reawakening of dormant bacteria and are postulated to be important for these processes. In this study, we explore the presence of dormant bacteria in the sputum of TB patients and further assess the ability of Rpf-containing and Rpf-deficient culture filtrates to stimulate growth of these organisms.
Methods: Sputum was obtained from 110 patients with culture positive drug sensitive pulmonary TB, before the initiation of treatment. Growth stimulation was monitored using most probable number (MPN) assays, conducted in the presence of Rpf-containing and Rpf deficient culture filtrates. All cultures were spoligotyped.
Results: In our patients, 32% of individuals carried Rpf-dependent differentially culturable TB (DCTB) populations. An additional 68% of individuals retained Rpf-independent DCTB. Within the latter group, 53% harbored populations which were suppressed by Rpf- containing filtrate. There was no statistically significant correlation between the presence of these DCTB populations with HIV status, CD4 count and the presence of pulmonary cavitations. The MPN assay allowed for TB detection in patients who reported either as smear negative, GeneXpert negative, MGIT negative or agar culture negative and facilitated the detection of mixed strain infections.
Conclusion: Our findings indicate that most individuals within our patient cohort harbour Rpf-dependent/Rpf-independent DCTB suggestive of great complexity in bacterial populations during TB infection. These observations have important implications for treatment response and outcome.
The contribution of Nth and Nei DNA glycosylases to mutagenesis in Mycobacterium smegmatis
Nabiela Moolla1, Vivianne J. Goosens1,2, Bavesh D. Kana1 and Bhavna G. Gordhan1 1
DST/NRF Centre of Excellence for Biomedical TB Research, School of Pathology, Faculty of Health Sciences, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg, South Africa; 2Department of Medical Microbiology, University of Groningen, University Medical Center Groningen, Hanzeplein 1, Groningen, The Netherlands.
Background: DNA damage is critical for mutagenesis in Mycobacterium tuberculosis, highlighting a critical role for DNA repair enzymes in mutation avoidance. Formamidopyrimidine (Fpg/MutM/Fapy) and EndonucleaseVIII (Nei) constitute the Fpg/Nei family of DNA glycosylases, which together with EndonucleaseIII (Nth), are central for DNA repair. In this study we assess the function of these enzymes in Mycobacterium smegmatis.
Methods: Homologues recombination was used for sequential inactivation of the nth, nei and fpg homologues to generate combinatorial mutants which were phenotypically characterized.
Results: Deletion of nth individually results in increased UV-induced mutagenesis and combinatorial deletion with the nei homologues results in reduced survival under oxidative stress conditions and an increase in spontaneous mutagenesis to rifampicin. Deletion of nth with the fpg homologues did not show any growth/survival defects or changes in mutation rate.
Conclusion: Collectively, our data confirm a role for Nth in DNA repair in mycobacteria and highlight a novel interplay between the Nth and Nei homologues in spontaneous mutagenesis and drug resistance in mycobacteria.
Characterization of the mycobacterial electron transport chain
Nicole Narrandes1 and Bavesh Kana1
1DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg, South Africa
Background: New interventions are required to combat the current TB epidemic. Pathways involved in energy generation, such as the electron transport chain (ETC) are hypothesized to play a critical role in the survival of mycobacteria.The ETC is responsible for maintaining an energized membrane, thereby facilitating the production of energy in the form of ATP and has recently been validated as a rich source for new drug targets.
Methods: Mutant strains defective for either the cytochrome bd oxidase (CbO) or the cytochrome bc-aa3 supercomplex (CcO) in the ETC were analysed for in vitro growth and ATP production.
Results: We show that in M. smegmatis CcO is essential for growth and is required for optimal ATP production in carbon rich media. Under nitrogen-limiting conditions, the CbO is required for optimal growth and ATP production. Loss of a putative CbO assembly protein in Mycobacterium tuberculosis hinders growth, whereas loss of the CbO appears to promote growth under carbon rich conditions.
Conclusion: These data highlight the flexibility inherent in the ETC and identify CbO as a potential new drug target whose inhibition will synergize with new emerging treatments.
How Flexible Can Mycobacterial Cell Walls Be? A Bioinformatic Approach.
Edith. Machowski1, Christopher Ealand1, Sibusisu Senzani1 and Bavesh Kana1
1DST/NRF Centre of Excellence for Biomedical TB Research, Faculty of Health Sciences, University of the Witwatersrand and the National Health Laboratory Service, Johannesburg, South Africa.
Background: Mycobacterium tuberculosis is able to lie dormant in the host for many years. One of the first lines of defense for any bacterium is the cell envelope. This in silico study investigated the repertoire of peptidoglycan (PG) remodelling genes, at publically accessible web sites utilising both raw and annotated data.
Methods: The chromosomal sequences of 19 mycobacterial strains were probed for the presence of genes encoding penicillin binding proteins, endopeptidases, L,D-transpeptidases, N-acetylmuramoyl-L-alanine amidases, and resuscitation promoting factors (Rpfs). Searches used amino acid sequences of proteins known to be involved in peptidoglycan remodelling.
Results: Phylogenetic analysis in this study confirmed the classification as Mycobacterium tuberculosis complex (MTBC), other pathogens, environmental strains and Mycobacterium leprae. Substantial genetic expansion in environmental strains is in contrast to contraction of the content M. leprae, as expected.
Conclusion: Mycobacterial petidoglycan remodelling enzymes display a great extent of variability, suggesting a functional role in allowing phenotypic diversification to allow the organisms to adapt.